ABSTRACT The goal of this study is to utilize PET imaging in GEMMs to perform a mechanistic study of mitochondrial heterogeneity following inactivation of the LKB1/AMPK signaling pathway during lung tumor development. LKB1 functions as a master kinase that regulates cellular energetics and mitochondrial function through activation of the adenosine monophosphate activated kinase (AMPK) that is frequently mutated in cancer. LKB1 mutations lead to inactivation of the AMPK signaling pathway resulting in severe defects in cellular energetics and mitochondrial homeostasis. This results in highly variable mitochondrial pools within human and mouse tumors that consist of numerous atypical mitochondria of differing size, morphology and function that we define as mitochondrial heterogeneity,. However, little is understood at a physiological or mechanistic level how mitochondrial heterogeneity resulting from LKB1 inactivation impact lung tumorigenesis or therapy. We examined mitochondrial structural and functional heterogeneity in lung tumors in vivo by coupling electron microscopy (EM) and positron emission tomography (PET) imaging of Lkb1-/- genetically engineered mouse models (GEMMs). Using a voltage sensitive mitochondrial specific radiotracer [18F]-Fluorobenzyl- triphenylphosphonium (FTP) we are able to measure mitochondrial membrane potential (??) in lung tumors by PET imaging. FTP PET imaging identified lung tumor populations with heterogeneous mitochondrial activity in vivo. Additionally, mitochondrial defects sensitize LKB1-/- tumor cells to undergo mitochondrial outer membrane permeabilization (MOMP) and apoptosis and we discovered the LKB1/AMPK pathway is a potential regulator of MOMP and apoptosis through voltage dependent anion 1 (VDAC1). Lastly, as a result of a synthetic lethal chemical screen, we identified protein tyrosine phosphatase mitochondria 1 (PTPMT1), a key regulator of cardiolipin biosynthesis and mitochondrial integrity as a novel therapeutic target in LKB1-/- lung cancer. We hypothesize that inactivation of the LKB1 tumor suppressor induces heterogeneity in mitochondrial structure and function that drives lung tumor development. To test this hypothesis we will integrate PET and EM imaging of Lkb1-/- GEMMs of lung cancer to longitudinally study mitochondrial heterogeneity at distinct stages of lung tumor development. In Aim1 we will use FTP PET imaging to map mitochondrial heterogeneity and dynamics in vivo during lung tumorigenesis following LKB1 loss. In Aim 2 we will identify the molecular mechanisms by which the LKB1/AMPK pathway regulates the mitochondrial outer membrane. In Aim 3 perform an in vivo dissection of the PTPMT1-cardiolipin pathway in LKB1-/- lung tumors. We propose first-in- field studies that will advance our fundamental understanding of mitochondrial biology and the impact of mitochondrial heterogeneity has on promoting lung tumorigenesis. The proposed work has relevance to human health in the areas of PET imaging based detection diagnosis of lung cancer as well as the development of new therapies to improve outcomes for lung cancer patients.